PROJECT SUMMARY/ABSTRACT Pain and pain sensitization are devastating side effects of tissue injury, cancer treatment, and certain diseases like diabetes. Treating pain effectively is an enormous clinical obstacle for many injuries and disease states. Pain research leaders have argued that new models in which novel conserved gene targets can be efficiently identified are urgently needed. Even more essential is the translation of new conserved targets functionally identified in these new and simple models to more clinically relevant vertebrate models. Our long-term goal is to identify novel pain/pain sensitization regulators with high potential for clinical translation. Over the last ten years my laboratory has performed a number of genetic screens in the fruit fly Drosophila, by combining unique tissue damage and pain assays. These screens have identified highly conserved regulators of pain biology (in particular acute and chronic pain sensitization following tissue damage) whose roles in regulating pain were not previously appreciated from work in other models. The two most promising screen hits as defined by conservation, robustness and uniqueness of their Drosophila phenotype, and novelty to pain biology, are Smoothened, the signal transducer of the Hh signaling pathway, and the Insulin receptor. In fly larvae, both genes are required in peripheral nociceptive sensory neurons? Smoothened to regulate acute thermal pain sensitization and the Insulin receptor (InR) to regulate the cessation of acute sensitization. InR is particularly interesting as the neuron-specific role in regulating the transition from acute to chronic sensitization may provide an entirely new way of looking at the pain associated with diabetes. Our goal in this exploratory R21 proposal is necessary and urgent. For both Smoothened and the Insulin Receptor we propose to perform parallel experiment(s) in mice to those that we have already performed in flies: we will conditionally knock these genes out in pain-sensing sensory neurons and assess baseline, acute, and chronic pain phenotypes in two standard assays. This is necessary because this type of forward translation of Drosophila-based results, though standard and useful in developmental biology and innate immunity, has not been previously attempted in any systematic way in the pain field. It is urgent because successful translation (meaning the genes do actually regulate pain biology in some way in mice) could establish a veritable pipeline of new conserved pain gene targets for testing in vertebrate models. This is especially true as we have many other conserved targets that have been identified in our screens. The results could be both scientifically compelling- providing new insight into conserved regulation of pain responses. They could also be clinically useful- pointing the way to new druggable conserved targets that have been difficult to identify systematically in other complementary experimental systems. The importance of pain-sensing and sensitizing mechanisms to the animal, reflected in the high evolutionary conservation of these mechanisms, suggest to us that this approach is likely to work.